RU2717339C2 - Application of infant formula containing sweet milk whey for provision of postnatal development of nervous system of baby's gastrointestinal tract and formation of intestinal functions, by which it controls - Google Patents

Abstract

FIELD: pediatrics.

SUBSTANCE: invention can be used for preventing or treating disorders associated with an immature or injured enteral nervous system in an infant or young child. That is ensured by using sweet milk whey protein (SWP), which is introduced into an infant or young child in a daily dose of 30–80 % of total protein intake.

EFFECT: invention provides developing enteral nervous system in infants, especially in premature and infants with low birth weight.

16 cl, 2 dwg, 3 tbl, 2 ex

Description

The scope of the invention

The present invention relates essentially to the field of nervous system health, protection of the nervous system and development of the nervous system. The invention, in particular, relates to the administration of whey protein to ensure the development of the enteric nervous system in infants, in particular in premature infants with low, very low and extremely low birth weight.

BACKGROUND OF THE INVENTION

The nervous system is a very complex network consisting of nerve and glial cells. It is present in all mammalian species and consists of the central nervous system (brain and spinal cord) and the peripheral nervous system (somatic, autonomous and enteric nervous system).

The central nervous system controls cognitive functions (memory, attention, perception, action, etc.). Together with the peripheral nervous system, it plays a fundamental role in controlling behavior. The somatic nervous system is responsible for the coordination of body movements (under the control of consciousness). The autonomic nervous system supports homeostasis in various types of body activity without consciousness control (heart rate, etc.). Finally, in the composition of the latter system, the enteric nervous system directly controls the functions of the gastrointestinal tract. They include the function of the gastrointestinal barrier and immune function, motility, absorption, digestion, and exocrine / endocrine secretion, which are involved in protecting the intestines from any type of damage and in ensuring digestion comfort [Neunlist, M. et al. (2008); Neuro-glial crosstalk in inflammatory bowel disease, J Intern. Med, 263: 577-583], [Burns, A.J. et al. (2009); Development of the enteric nervous system and its role in intestinal motility during fetal and early postnatal stages, Semin. Pediatr. Surg., 18: 196-205], [Tapper, E.J. (1983); Local modulation of intestinal ion transport by enteric neurons, Am J Physiol., 244: G457-68].

The nervous system develops during pregnancy, and then finally forms into a mature functional network in the postnatal period.

Due to the crucial role of the enteric nervous system in the functioning of the gastrointestinal tract, immaturity or delayed maturation of this nervous system leads to a delay in the formation and proper functioning of the basic functions of the gastrointestinal tract. In particular, it contributes to:

- immaturity of motility of the gastrointestinal tract [Burns, A.J. et al. (2009); Development of the enteric nervous system and its role in intestinal motility during fetal and early postnatal stages, Semin. Pediatr. Surg., 18: 196-205], which leads to delayed intestinal transit with harder stools and causes discomfort in the intestines, and in more severe cases leads to constipation, to an increased susceptibility of the infant to intolerance to enteral nutrition, the need for total parenteral nutrition and , in more severe cases, necrotizing enterocolitis (NEC) (Grave GD, Nelson SA, Walker WA, Moss RL, Dvorak B, Hamilton FA, Higgins R, Raju TN. New therapies and preventive approaches for necrotizing enterocolitis: report of a research planning workshop Pediatr Res. 2007 Oct; 62 (4): 510-4; Indrio F, Riezzo G, Cavallo L, Di Mauro A, Francavilla R. Physiological basis of food intolerance in VLBW. J Maternal Fetal Neonatal Med. 2011 Oct; 24 Suppl 1: 64-6;

- reduced digestive / absorption capacity of the intestine [Tapper, E.J. (1983); Local modulation of intestinal ion transport by enteric neurons, Am J Physiol., 244: G457-68]; Josef Neu & Liyan Zhang, Feeding intolerance in very low birth weight infants: What is it and what can we do about it? Acta Paediatrica 2005; 94 (suppl 449): 93-99];

- immaturity of the barrier function of the intestine [Neunlist, M. et al. (2008); Neuroglial crosstalk in inflammatory bowel disease, J Intern. Med, 263: 577-583, Burns, A.J. et al. (2009)], which increases the risks of infections, intolerance to enteral nutrition and necrotizing enterocolitis (NEC) [Athalye-Jape G, More K, Patole S Progress in the field of necrotising enterocolitis-year 2012 .. J Maternal Fetal Neonatal Med. 2013 May; 26 (7): 625-32; Indrio F, Riezzo G, Cavallo L, Di Mauro A, Francavilla R. Physiological basis of food intolerance in VLBW. J Maternal Fetal Neonatal Med. 2011 Oct; 24 Suppl 1: 64-6].

An immature or damaged enteric nervous system can be observed in the babies listed below.

- Premature babies, infants with low birth weight (<2500 g), very low birth weight (<1500 g) and extremely low birth weight (<1000 g). New J. Gastrointestinal development and meeting the nutritional needs of premature infants Am J Clin Nutr 2007; 85 (2): 629S-634S.

, M.C.S. et al., (2003); Effects of pre- and postnatal protein energy deprivation on the myenteric plexus of the small intestine: a morphometric study in weanling rats, Nutr. Res., 23: 215-223].- Infants born prematurely or on time, with intrauterine growth retardation (IUGR), which occurred as a result of any undesirable effects during pregnancy (maternal smoking, mother taking medications, poor placenta quality, improper placenta placement, improper placentation, and maternal nutrition and fetus, excessive stress / anxiety of the mother, etc.). [Shanklin DR and Cooke RJ (1993); Effects of intrauterine growth on intestinal length in the human foetus, Biol Neonate, 64: 76-81], [Neu, J. (2007); Gastrointestinal development and meeting the nutritional needs of premature infants, Am. J. Clin. Nutr., 85 (2): 629S-634S], [

, MCS et al., (2003); Effects of pre- and postnatal protein energy deprivation on the myenteric plexus of the small intestine: a morphometric study in weanling rats, Nutr. Res., 23: 215-223].

- Any newborn child and an infant with a delay in the development of the nervous system as a result of, for example, hypoxemia-ischemia at birth or any other undesirable phenomenon [Taylor, S.T. and Colgan S.P. (2007); Hypoxia and gastrointestinal disease, J. Mol. Med. (Berl.), 85: 1295-300], [Barrett R. D. et al. (2007); Destruction and reconstruction: hypoxia and the developing brain, Birth Defects Res With Embryo Today, 81: 163-76].

- Any newborn child and infant with impaired gastrointestinal function (digestive disorders, motility disorders, gastrointestinal reflux, slow gastrointestinal transit, intolerance to oral feeding), Hirschsprung's disease and inflammation that encompasses the gastrointestinal tract (such as necrotizing enterocolitis), and obstructive pathological conditions [Burns AJ et al. (2009); Development of the enteric nervous system and its role in intestinal motility during fetal and early postnatal stages, Semin. Pediatr Surg., 18 (4): 196-205].

In humans, neural crest cells, from which the enteric nervous system is formed, are known to develop in the fetus in utero shortly after conception (starting at 7.5 weeks of development) [Burns, A. J. and Thapar, N. (2006); Advances in ontogeny of the enteric nervous system, Neurogastroenterol. Motil., 18, 876-887]. It has been shown that the enteric nervous system undergoes significant changes after birth until the age of six years, while less significant changes continue up to ten years [Wester, T. et al. (1999); Notable postnatal alterations in the myenteric plexus of normal human bowel, Gut, 44: 666-674]. Thus, if the fetus, newborn child or infant has a delay in the development of the nervous system, it is desirable to quickly reverse this delay so that the development of the nervous system “catches up” to a normal level. It is desirable that any damage to the enteric nervous system be repaired as soon as possible, so that the growing fetus or infant has as few disorders of the gastrointestinal tract or other pathological conditions associated with an immature or damaged enteric nervous system.

Thus, the healthy development of the enteric nervous system in the fetus, newborn, and growing child helps to manage the proper formation and preservation of intestinal motility, intestinal barrier function, and thus the intestinal absorption and digestive functions. This helps prevent inflammatory pathological conditions associated with impaired bowel function, and also reduces the risk of infection and allergies (Neunlist et al., 2008).

Thus, peri- and / or postnatal interventions are in line with a promising approach to ensure the healthy development of the enteric nervous system. Pregnancy / lactation interventions can have significant advantages in terms of convenience and compliance compared to interventions designed for use in the baby.

There is a need to ensure and maintain healthy development and / or restoration of the enteric nervous system in the very early stages of pregnancy, as well as in the early stages of a newborn’s life during the rapid maturation of the nervous system. Since the nervous system continues to develop during the first years of childhood (up to the age of about ten years), during this period such a need for constant support is relevant.

In particular, there is a need to support the healthy development of the enteric nervous system in newborns, infants and young children in order to best prepare them for stresses in the gastrointestinal tract, such as changes in diet, chemical (e.g. drugs) or physical (friction) ) damage, infections, inflammatory / immune reactions, etc., as well as to improve the future maturation of their enteric nervous system in later life.

There is a need to provide such support or such an appropriate composition in a form that is acceptable to a population of subjects, in particular to those individuals in these populations who are most vulnerable or most in need. In addition, there is a need to not cause adverse events, side or negative effects in such a population. There is a need to provide such solutions for populations of subjects in the simplest and most cost-effective way.

The present invention relates to all mammals, including animals and humans.

SUMMARY OF THE INVENTION

The invention relates to the use of sweet whey protein (SWP) to ensure healthy development and / or restoration of the enteric nervous system in newborns. The sweet whey protein may be a modified sweet whey protein (MSWP) from which caseinoglycomacropeptide (CGMP) has been removed. SWP can be partially or substantially hydrolyzed.

The introduction of sweet whey protein ensures the normal and healthy development of nerve and glial cells in the intestine. It also provides healthy differentiation of neurons in the peripheral nervous system. Thus, the introduction of SWP in accordance with the invention allows to prevent and treat disorders associated with immature or damaged enteric nervous system. The introduction of SWP in accordance with the invention provides healthy development and / or restoration of the enteric nervous system in young mammals.

The disorder may be a violation of the barrier function of the intestine, food intolerance, or necrotizing enterocolitis. The disorder may be a disorder that is directly related to impaired intestinal barrier function. In a more general case, the disorder may be a violation of the motor function of the gastrointestinal tract, which may manifest as slow intestinal transit, intestinal discomfort, hard stool, constipation and / or gastrointestinal reflux.

The introduction of sweet whey protein to the fetus can be done through the mother. Administration can also be done to a premature or full-term infant directly or through breast milk. Administration to a child can also be carried out substantially up to the age of six years or to an animal of equivalent age.

The sweet whey protein can be administered to an infant or a baby starting to walk, directly in its pure form or diluted with water or breast milk, in the form of a biologically active food supplement (BAA) or together with a milk fortifier, or with any milk supplement used in nutritional time, in infant formula, or in a milk-based drink. Protein sweet whey is administered to an infant or young child in a daily dose of 30-80%, preferably 60 wt. % of total protein intake.

The administration period for the fetus is essentially at least one week, preferably two weeks, more preferably at least one month, and the administration period for the infant or young child is essentially at least 4 weeks, preferably 2-12 months, more preferably at least 18 months, and even more preferably up to the age of 6 years.

MSWP can be administered to a premature or full-term infant or child or young adult in a dose of 1.6-3.2 g protein / 100 kcal, preferably 1.6-2.2 g protein / 100 kcal, and more preferably 1.8 -2.1 g protein / 100 kcal. In one embodiment, MSWP is administered to the infant at a dose of 1.0 to less than 1.6 g protein / 100 kcal.

The invention relates to a composition containing 30-80%, preferably 60% sweet whey protein for the prevention or treatment of disorders associated with immature or damaged enteral nervous system in a young mammal.

The invention relates to the use of SWT, and more particularly to compositions containing 30-80 wt. % SWP, to ensure healthy development and / or restoration of the enteric nervous system in young mammals.

Brief Description of the Drawings

FIG. 1. Stimulation of the contractile reaction ex-vivo in the jejunum by an electric field.

по сравнению с PR-W. Сокращения означают следующее: PR - ограничение белка; W - вода; MSWP28 - модифицированная сладкая молочная сыворотка Р28; концентрат GF - концентрат ростовых факторов.Stresses (area under the curve (AUC)) obtained by isometric contractions of the jejunum in response to stimulation by an electric field at 10 Hz in CTRL-w, PR-w, and PR cubs to which various milk fractions measured after sacrifice were added (14th day after birth). The results are medians ± st. osh medians, n = 6-10 depending on the group, P <0.05; * compared to CTRL-W and

compared to PR-W. Abbreviations mean the following: PR - protein restriction; W is water; MSWP28 - modified sweet whey P28; GF concentrate - concentrate of growth factors.

FIG. 2. Stimulation of ex-vivo contractile reaction in the jejunum by acetylcholine.

по сравнению с PR-W. Сокращения означают следующее: PR - ограничение белка; W - вода; MSWP28 - модифицированная сладкая молочная сыворотка Р28; концентрат GF - концентрат ростовых факторов.Stresses (area under the curve (AUC)) obtained by isometric contractions of the jejunum in response to stimulation with 10 ^-6 M acetylcholine stimulation in CTRL-w, PR-w, and PR pups to which various milk fractions measured after sacrifice were added (14 day after birth). The results are medians ± st. osh medians, n = 6-10 depending on the group, P <0.05; * compared to CTRL-W and

compared to PR-W. Abbreviations mean the following: PR - protein restriction; W is water; MSWP28 - modified sweet whey P28; GF concentrate - concentrate of growth factors.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In this description, the following terms have the following meanings.

“Infants”: in accordance with EU Commission Directive 2006/141 / EC of 22 December 2006 on infant formula and infant formula for nursing infants, paragraph 1.2 (a), the term “infants” means infants under 12 months of age.

“Newborn baby” essentially means a baby under the age of 6 months.

"Premature infant" essentially means an infant born before 37 weeks of pregnancy.

“Full-term infant” essentially means an infant born after 37 weeks of gestation.

“A child starting to walk” essentially means a child from the moment he learned to walk to three years.

"Young child" essentially means a child aged from one to ten years (and at an equivalent age in animals, if the term "young mammals" is used).

“Modified sweet whey protein” (MSWP) or “modified sweet whey” (MSW) means a sweet whey protein from which part or all of the caseinoglycomacropeptide (CGMP) has been removed. In various embodiments, more than 60%, more than 75%, more than 90%, more than 95%, or more than 99 wt.% Are removed from the MSW. % caseinoglycomacropeptide (CGMP) compared to the average amount present in native sweet whey of the same origin (for example, cow's sweet whey).

The term "probiotic" means preparations from microorganism cells or components of microorganism cells that have a beneficial effect on the health or condition of the host organism. [Salminen, S. et al. (1999); Probiotics: how should they be defined Trends Food Sci. Technol., 10 107-10]. The definition of a probiotic is essentially universally accepted and consistent with the definition of WHO. A probiotic may contain a unique strain of a microorganism, a mixture of different strains and / or a mixture of different species and genera of bacteria. In the case of mixtures, the term “probiotic” in the singular can still be used to refer to a mixture of probiotics or a preparation. For the purpose of the present invention, microorganisms of the genus Lactobacillus are considered probiotics.

“Prebiotic” essentially means a non-digestible food ingredient that favorably affects the host organism by selectively stimulating the growth and / or activity of microorganisms present in the intestines of the host organism and, therefore, attempts to improve the health status of the host organism.

“Allergy” means an allergy that has been identified by a doctor and that can be treated sporadically or for longer. "Food Allergy" means an allergy associated with a nutritional composition.

“Baby formula”: in accordance with EU Commission Directives 2006/141 / EC of December 22, 2006 and / or 91/321 / EEC of May 14, 1991 on infant formula and infant formula, paragraph 1.2 (c) , the term "infant formula" means food products intended for special use in food by infants in the first four to six months of life, which themselves satisfy the nutritional needs of this category of people. It must be understood that babies can be fed exclusively with infant formula, or the carer can use the infant formula as a supplement to breast milk. It is synonymous with the widespread expression "initial infant formula."

“Mixtures for nursing infants”: in accordance with EU Commission Directives 2006/141 / EC of 22 December 2006 and / or 91/321 / EEC of May 14, 1991 on infant mixtures and mixtures for nursing infants, paragraph 1.2 ( d), the term “formula for nursing babies” means foods intended for special use in food by infants older than four months and constituting the main liquid component in the diet with a gradually increasing variety of diets for this category of people.

“Milk formula for children 1-3 years old”: a nutritional composition based on milk, specially adapted for a child aged from one to three years.

“Breast milk fortifier”: a nutritional composition for infants or young children to be added to breast milk or diluted with breast milk.

The term “hypoallergenic composition” means a composition that is less likely to cause allergic reactions.

The term "sialylated oligosaccharide" means an oligosaccharide having a sialic acid residue.

The term “fucosylated oligosaccharide” means an oligosaccharide having a fucose residue.

Unless otherwise specified, all percentages given are given as percentages by weight.

When used in this description, the words "contains", "comprising" and similar words should not be interpreted in an exclusive or comprehensive sense. In other words, it is assumed that they mean "including, without limitation."

Any reference to documents of the prior art in this description should not be construed as recognition that such prior art is widely known or forms part of well-known knowledge in the field.

Dietary protein provides the essential amino acids needed for protein synthesis and growth, and protein quality is as important as the amount of protein. The present invention provides a sweet whey protein for administration by administration in order to ensure the healthy development of the enteric nervous system in mammals. Sweet whey can be obtained in the manufacture of cheese, in particular, sweet whey is obtained after coagulation of casein with rennet.

Protein sweet whey in accordance with the invention contains 30-100 wt. % protein, preferably> 80 wt. % squirrel. An example of a SWP that can be used in accordance with the invention is the SWP sold under the trademark Lacprodan Di9224 ™. Thus, the SWP may be in the form of a whey protein isolate or a whey protein concentrate.

According to one embodiment of the invention, the sweet whey protein is a fraction of the modified sweet whey, that is, the sweet whey protein with a reduced level of caseinoglycomacropeptide (CGMP) compared to the classic sweet whey protein. This fraction of sweet whey is called “modified sweet whey” (MSW). Classic sweet whey may contain from 4 to 40% CGMP, depending on how the milk is processed.

The protein fraction in cow's milk is a mixture of several proteins, all of which have different amino acid profiles. Caseinoglycomacropeptide (CGMP) is obtained by proteolysis of kappa-casein to form para-kappa-casein, an insoluble fraction that remains in the casein fraction, and CGMP, a soluble fraction that is in the whey fraction. This fraction of whey with reduced CGMP, or MSW, provides the advantage of reduced threonine and increased tryptophan content over conventional sweet whey and is therefore suitable as a protein source for infants.

This fraction of sweet whey can be further processed to remove minerals (cations, anions), lactose, or any of these substances. If desired, sweet whey can be concentrated. Suitable sources of sweet whey are commercially available.

Removal of caseinoglycomacropeptide can be accomplished by any suitable method. One suitable method is described in EP0880902. In this method, the pH of the sweet whey is adjusted to 1-4.3 if necessary. Then, sweet whey is contacted with a weakly anionic resin, which is predominantly alkaline, until the pH of the sweet whey stabilizes at about 4.5-5.5. After that, a fraction of sweet whey was collected, from which a significant amount of caseinoglycomacropeptide was removed. In accordance with one embodiment of the invention, such a reduced CGMP whey protein fraction contains about 28% protein, of which CGMP is 2-3% of the total protein, and therefore is called MSWP28.

An MSW intended for use in accordance with the invention may, of course, contain a higher percentage of protein than MSWP28, for example 30-99% protein. Similarly, an MSW intended for use in accordance with the invention may also contain a higher percentage of CGMP than MSWP28, for example up to 15, 20, 25, or 35% of the total protein.

According to another embodiment of the invention, the sweet whey protein is not modified and comprises at least 50% of the protein. For example, SWP sold under the trademark Lacprodan Di9224 ™ by Aria Foods Ingredients, Skanderborgvej 277, 8260 Viby J, Denmark, contains 87% protein.

Other examples of commercially available unmodified sweet whey protein for use in accordance with the invention are Lacprodan DI-8790, another sweet whey protein from Aria, 894 Instantised and MPC 485, put into serial production by Fonterra (New Zealand), YV0608, Armor Proéines (France) and BiPro, Davisco Food International inc (USA, 11000 West 78th Street, Suite 210, Eden Prairie, MN 55344).

Sweet whey protein can be non-hydrolyzed. Alternatively, in some embodiments, the whey protein fraction can be partially or substantially hydrolyzed to prevent allergic reactions in infants at risk of developing allergies, and to facilitate protein digestion. The hydrolysis process can be carried out at will and as is known in the art. Essentially a whey protein hydrolyzate is produced by enzymatically hydrolyzing a fraction of sweet whey in one or more steps. For example, to obtain a substantially hydrolyzed protein, sweet whey proteins can be triple hydrolyzed using, for example, 2.4 L alkalase (EC 940459), then 0.5 L neutralase (available from Novo Nordisk Ferment AG), and then pancreatin at 55 ° C. Alternatively, to obtain a less hydrolyzed protein fraction, sweet whey can be double hydrolyzed using, for example, NOVOZYMES enzymes and then pancreatin.

It has been established that if the sweet whey protein used is essentially free of lactose, then the protein undergoes lysine blockade to a much lesser extent during hydrolysis. This allows to reduce the degree of blockade of lysine from approximately 15 wt. % total lysine to less than about 10 wt. % lysine; for example, about 7 wt. % lysine. This significantly improves the nutritional quality of the protein source.

The introduction of SWP to the fetus can be done through the mother. Administration can also be done to a premature or full-term infant directly or through breast milk. The introduction can also be carried out to a young child essentially up to the age of four years or an animal of equivalent age.

The introduction of SWP to a young mammal, which may be a human (fetus, infant, baby, beginner, or young child) or animal, has a positive effect on the development of their enteric nervous system, which allows this system to mature and develop normally. Thus, the administration of a sweet whey protein in accordance with the invention allows the prevention and treatment of disorders associated with an immature or damaged enteric nervous system.

These disorders can be, for example, impaired intestinal barrier function, impaired intestinal barrier function, or necrotizing enterocolitis. The latter conditions increase the risk of infection and allergies, which leads to food intolerance (hence the need for parenteral nutritional support). More generally, these disorders may constitute a violation of the motor function of the gastrointestinal tract, which may manifest as slow intestinal transit, intestinal discomfort, hard stool, constipation and / or gastrointestinal reflux.

The beneficial effects of the invention are particularly directed to those young mammals that have experienced, for example, intrauterine growth retardation (IUGR), which may occur after any adverse event during pregnancy (e.g., active or passive smoking of the mother, medication by the mother, low quality placenta , improper location of the placenta, malnutrition of the mother and / or fetus, etc.).

The inventors of the present invention have found that SWP and / or SWP-containing compositions of the present invention can be used to provide for the development, functionality, survival, plasticity and differentiation of neurons and to protect neurons from degeneration, as demonstrated by stimulating the expression of proteins associated with these types of biological activity. Such degeneration can develop after, for example, any stressful situations, such as situations affecting the fetus (in utero), for example, the aforementioned IUGR, or in newborns (hypoxia-ischemia at birth, oxygen therapy and hyperoxia, inflammation, the need for parenteral support, etc.), or any cause leading to oxidative stress. It was found that SWP ensures the survival of neurons and / or limits or prevents the death of enteric neurons, and also ensures the development of the nervous system, which, for example, is important for developmental processes.

In infants, SWPs and / or compositions containing SWPs in accordance with the present invention can be used to protect the enteric nervous system from any stress, such as occurring during the development of the nervous system, and therefore to limit and / or prevent the stress-induced delay in the development of the nervous system and corresponding intestinal dysfunction.

Thus, in the context of the present invention, SWP can be administered already after detecting a delay in the development of the enteric nervous system or for prevention, when such a delay is not yet observed.

The beneficial effects of SWP on the healthy development of the intestinal nervous system in a mammal are described in more detail in the paragraphs below.

Doses of SWP

Sweet whey protein can be administered to an infant or young child at a dose of 1.6-3.2 g protein / 100 kcal, preferably 1.6-2.2 g protein / 100 kcal, and even more preferably 1.8-2.1 g protein / 100 kcal.

There are special recommendations published by the ESPGHAN Nutrition Committee for premature babies on the amount of protein they need to receive. In the case of premature babies born with a weight of less than 1 kg, the recommended protein intake is from 3.6 to 4.1 g of protein / 100 kcal. For infants born with a weight of 1 to 1.8 kg, the recommended protein intake is from 3.2 to 3.6 g protein / 100 kcal [Agostini et al. (2010) JPGN 2010 (50), 1, Enteral Nutrient Supply for Preterm Infants].

Thus, the amount of SWP administered to a premature infant is appropriately adapted in accordance with current recommendations. For example, if, in accordance with one embodiment of the invention, the SWP contains 100% protein and the SWP is 80% of the total protein administered to the premature baby, then the appropriate amount of SWP to be administered to the premature baby is 2.8-3.2 g per 100 kcal for infants with a body weight of less than 1 kg and 2.5-2.9 for infants with a body weight of 1 kg to 1.8 kg.

The administered dose of SWP should be such that the protein intake of the subject is within the appropriate guidelines (e.g., WHO recommendations or ESPGHAN committee).

For example, in a preferred embodiment, the composition comprises from about 9.0 to about 10.0 wt. % protein, more preferably about 9.5 wt. % This corresponds to approximately 1.8 g of protein / 100 kcal. The advantage provided by this concentration of protein is that it is equivalent to the amount of protein essentially present in breast milk and corresponds to the lower limit described in the Codex Alimentarius.

Essentially, MSWP may comprise from about 70 to about 100% of the total protein in the composition. Thus, it can be 75%, 80%, 85%, 90% or 95% of the protein in the composition.

Thus, the modified sweet whey protein in accordance with the invention can essentially be administered to an infant or young child at a dose of 1.6-3.2 g protein / 100 kcal, preferably 1.6-2.2 g protein / 100 kcal and even more preferably 1.8-2.1 g of protein / 100 kcal.

Route of administration

(i) Introduction to infants

SWP can be administered separately to infants directly orally (for example, in pure form or diluted with water or breast milk) as a biologically active food supplement (dietary supplement) (for example, as an additive in or with breast milk fortifier), or with any dairy supplement used during nutrition, either as a pharmaceutical or nutraceutical composition, or as an ingredient in infant formula. Such a mixture may be a “mixture for premature babies” if the baby was born prematurely or with a low birth weight, “an initial infant formula” or “a mixture for nursing infants”. The infant formula may also be a hypoallergenic (HA) infant formula in which cow's milk proteins are hydrolyzed. An example of such an initial infant formula is given in Example 2. SWP can be administered as a milk formula for children 1-3 years old or in any milk-based drink.

(ii) Introduction to young children

SWP can also be administered orally to young children in the form of a pharmaceutical or nutraceutical composition, an infant formula for children 1-3 years old, milk-based drinks, dietary supplements (BAA), milk yoghurts, desserts and puddings, cookies and grains bars, cereals and fruit drinks.

(iii) Administration to pregnant or nursing mothers

SWP can also be administered orally to pregnant or lactating mothers, preferably via food, drink, dietary supplements, or pharmaceutical compositions.

(iv) Introduction to animals

SWP can also be administered orally to animals alone, or in water, or in the form of a biologically active food supplement (BAA), pharmaceutical or nutraceutical composition, or milk, or animal feed.

Introduction with other compounds

SWP can be administered alone (for example, in pure form or diluted with water or milk, including breast milk) or in a mixture with other compounds (such as dietary supplements, nutritional supplements, drugs, carriers, flavors, digestible or non-digestible ingredients). Vitamins and minerals are examples of typical dietary supplements. In a preferred embodiment, SWP is administered in compositions, for example, infant formula, together with other compounds that enhance the described beneficial effect on young mammals. For example, it may be a probiotic.

Other probiotics may also be administered. Preferably, for this purpose, the probiotic can be selected from the group consisting of Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Kluyveromyces, Saccharoymces, Candida, in particular, can be selected from the group consisting of Bifidobacterium bifidobiferium bactidium bactidium bactidium bactidium bis bacterium lactis Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillusparacasei, Lactobacillus salivarius, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus salivarius, Lactococcus lactis, Enterococcus faecium, Saccharomyces cerevisiae, Saccharomyces boulardii or mixtures thereof, may preferably be selected from the group consisting of Bifidobacterium longum NCC3001 (ATCC BAA-999), Bifid obacterium longum NCC2705 (CNCM I-2618), Bifidobacterium longum NCC490 (CNCM I-2170), Bifidobacterium lactis NCC2818 (CNCM I-3446), Bifidobacterium breve strain A, Lactobacillus paracasei NCC2461 (CNCM Lactii Imoni161) I-1225), Lactobacillus rhamnosus GG (ATCC53103), Lactobacillus rhamnosus NCC4007 (CGMCC 1.3724), Enterococcus faecium SF 68 (NCC2768; NCIMB10415) and mixtures thereof.

Other examples of synergistic compounds that can be included in compositions, especially in the infant formula of the invention, are prebiotic compounds. A prebiotic is a non-digestible food ingredient that favorably affects the host organism by selectively stimulating the growth and / or activity of one or a limited number of bacteria in the large intestine and thus improves the health status of the host organism. Such ingredients are indigestible in the sense that they are not broken down and absorbed in the stomach or small intestine and thus, intactly pass into the large intestine, in which they are selectively fermented by beneficial bacteria. Examples of prebiotics include certain oligosaccharides, such as fructooligosaccharides (FOS), cow's milk oligosaccharides (CMOS), and galactooligosaccharides (GOS). A combination of prebiotics such as 90% GOS and 10% short chain fructooligosaccharides, for example, as a product sold under the Raftilose® trademark, or 10% inulin, for example, as a product sold under the Raftiline® trademark, can be used. Other examples of prebiotics that can be used in the context of the present invention include a group of oligosaccharides derived from milk or other sources optionally containing sialic acid, fructose, fucose, galactose or mannose. Preferred prebiotics are sialooligosaccharides (SOS), fructooligosaccharides (FOS), galactooligosaccharides (IMO), xyloligosaccharides (XOS), arabinoxyl oligosaccharides, AXOS), mannanosyl oligosaccharides, LS , sialyllactose (SL), fucosyllactose (FL), lacto-N-neotetraose (LNNT), lactulose (LA), palatinose oligosaccharides (RAO), maltooligosaccharides, resins and / or their hydrolysates, pectins, starches and / or their hydrolysates. The infant formula in accordance with the invention preferably further comprises at least one prebiotic in an amount of 0.3 to 10% of the total weight of the dry composition.

In particular, breast milk oligosaccharides, for example the sialylated oligosaccharides described in WO 2012/069416, published May 31, 2012, can be included in the composition in accordance with the invention. The latter oligosaccharides can act synergistically with the SWP of the invention to ensure the healthy development of the mammalian enteric nervous system in an infant or young child.

The daily doses of carbohydrates and all other compounds administered with SWP must in any case be in accordance with the published safety guidelines and regulatory requirements. This is especially important when administered to newborns, in particular children with low birth weight, very low or extremely low birth weight.

A composition, for example, an infant formula containing SWP for administration in accordance with one embodiment of the invention, may contain an additional protein source in such an amount that the total protein content is not more than 4.0, 3.0 or 2.0 g / 100 kcal, preferably 1.8 to 2.0 g / 100 kcal. Preferably, more than 50 wt. % of the protein source was whey, which includes modified sweet whey (MSW) and / or unmodified sweet whey. The type of additional protein source is not considered important for the present invention, provided that the minimum requirements for the content of essential amino acids are met and satisfactory growth is ensured. In one embodiment, the protein content is from 30% to 80% of whey proteins. Thus, additional protein sources such as skim milk, casein or soy can be used. In one embodiment, the casein / whey ratio is from 70/30 to 20/80.

Proteins may be intact or hydrolyzed, or may be a mixture of intact and hydrolyzed proteins. It may be desirable to administer partially hydrolyzed proteins (degree of hydrolysis of 2 to 20%), for example, to infants who are at risk of developing an allergy to cow's milk. If necessary, the use of hydrolyzed proteins, the hydrolysis process can be carried out at will and as is known in this field.

The composition may also contain a source of carbohydrates and / or a source of fat. The infant formula may contain a source of lipids. The lipid source may be any lipid or fat that is suitable for use in infant formulas. Preferred sources of fat include palm oil, high oleic acid sunflower oil and high oleic acid safflower oil. Essential fatty acids, linoleic and α-linolenic acid can also be added. One or more essential long chain fatty acids (DC-PUFA) may be included in the composition. Examples of DC-PUFA that can be added are docosahexaenoic acid (DHA) and arachidonic acid (AA). DC-PUFAs can be added at concentrations that comprise more than 0.01% of the fatty acids present in the composition. They can be added as small amounts of oils containing large amounts of pre-prepared arachidonic acid and docosahexaenoic acid, such as fish oils or microbial oils. Palmitic acid can be added, preferably at the Sn-2 position. In general, the fat content should be preferably such as to provide 30-55% of the total energy value of the infant formula. The fat source preferably has an n-6 to n-3 fatty acid ratio of from about 5: 1 to about 15: 1; for example from about 8: 1 to about 10: 1.

An additional source of carbohydrates may be added to the nutritional composition. Preferably, it should provide from about 40% to about 80% of the energy value of the nutritional composition. Any suitable carbohydrate may be used, for example, sucrose, lactose, glucose, fructose, dry corn syrup, maltodextrin, or a mixture thereof.

If desired, additional dietary fiber can also be added. In the case of addition, preferably it should be up to about 5% of the energy value of the nutritional composition. Additional dietary fiber can be obtained from any suitable source, including, for example, soy, peas, oats, pectin, guar gum, Arabian gum, oligosaccharides, including the FOS, GOS and oligosaccharides described above, or a mixture thereof. Suitable vitamins and minerals may be included in the nutritional composition in an amount consistent with the guidelines.

Examples of minerals, vitamins, and other nutrients optionally present in the infant formula include Vitamin A, Vitamin B1, Vitamin B2, Vitamin B6, Vitamin B12, Vitamin E, Vitamin K, Vitamin C, Vitamin D, Folic Acid, Inositol , niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chloride, potassium, sodium, selenium, chromium, molybdenum, taurine and L-carnitine. Minerals are usually added in salt form. The presence and quantity of specific minerals and other vitamins depends on the target population of infants.

The infant formula may optionally contain other substances that may have a beneficial effect, such as fibers, lactoferrin, nucleotides, nucleosides, and the like.

If desired, one or more edible emulsifiers can be included in the nutritional composition, for example, diacetyl tartaric acid esters of mono- and diglycerides, lecithin and mono- or diglycerides, or a mixture thereof. Similarly suitable salts and / or stabilizers may be included. Flavors may be added to the composition.

Administration period

The duration of administration may vary. Although positive effects are expected over a relatively short period of administration (e.g., daily administration to newborns for one to two weeks), with longer administration, the effect is expected to be stronger or at least persist in older infants (e.g., duration administration of three, five, eight or 12 months) or in children (for example, a duration of administration of up to 2, or 4, or 6 years). When administered to animals, the appropriate duration of administration is used.

A pregnant mother can start taking a modified sweet whey protein or a standard (unmodified) sweet whey protein as soon as she finds out about her pregnancy. However, the introduction can also begin before pregnancy, for example, if a woman wants to become pregnant. The introduction can be started at any time after the onset of pregnancy. It can be started preferably at the 3rd, 4th, 5th, 6th, 7th or 8th month of pregnancy in case of pregnancy in humans, or at appropriate periods for other mammals, or no later than two weeks before the expected date of birth.

The period of administration can be continuous (for example, before and including lactation, and even weaning) or intermittent. Continuous administration is preferred due to the development of a more sustainable effect. However, it is contemplated that intermittent administration (eg, daily administration for one week per month or every other week) may have a positive effect on the offspring.

Administration may cover at least a portion of the gestation period and at least a portion of the lactation period or an equivalent period if the newborn is not breastfed. Preferably, the administration period for the pregnant mother covers substantially the entire pregnancy period, although it may be less. Similarly, the administration period for the nursing mother preferably covers substantially the entire lactation period, although, again, this period may be shorter.

Preferably, the introduction of the mother is carried out by daily ingestion (once or twice a day) or weekly ingestion (once or twice a week).

Modified sweet whey protein or standard sweet whey protein can be administered directly to the infant. In particular, this is relevant if the mother does not breast-feed or when she stops breast-feeding. However, a breast-fed infant may also receive a modified sweet whey protein or a standard sweet whey protein by direct administration.

Preferably, administration to the infant is by daily oral administration. For example, if a modified sweet whey protein or sweet whey protein is administered in an infant formula, administration is carried out during each feeding, i.e. from about four to about six times a day for infants, and the number of feeds decreases with age. In young children (older than one year), administration can be carried out less often, once or twice a day. In children starting to walk, and children up to the age of six, the introduction is carried out if the use of milk is recommended for growth and development.

Administration to the infant by either breastfeeding or direct administration, or both, can be continued for up to six months, or even one year, or longer. Thus, a modified sweet whey protein or a standard sweet whey protein can be administered during lactation, if there is lactation, or after partial or complete weaning. The introduction can be continued at a time when the child begins to walk, and even up to the age of six years. The enteral nervous system is known to continue to develop in children up to this age [Wester, T. et al. (1999); Notable postnatal alterations in the myenteric plexus of normal human bowel, Gut, 44: 666-674]. Thus, the authors of the present invention suggest that the introduction of a modified protein of sweet whey or standard protein of sweet whey can be continued to obtain a positive effect, usually up to the age of six years.

Impact of introducing MSWP or standard SWP

Protein modified sweet whey or standard sweet whey, which is administered to newborns, provides a healthy development of the enteric nervous system. In an experiment with a rat model described in detail in Example 1, the effect of the administration of modified sweet whey (MSWP28) or a commercially available sweet whey protein (Lacprodan Di9224) on the development of the nervous system was evaluated.

In this experiment, calves with intrauterine growth retardation induced by the mother’s diet (PR group) and calves who did not have IUGR (CTRL) were given water as an additive from the 2nd day after birth (control animals; namely, CTRL-w and PR-w) or one of the following fractions of milk:

- Lacprodan Di9224: whey protein isolate, sweet whey, put into serial production by Arla Foods Ingredients, the Netherlands;

- MSWP28: sweet whey modified using the patented Nestle method (cGMP removal);

- GF concentrate: a concentrate of growth factors, which is an isolate with a high protein content (97% protein), extracted directly from skim milk and put into serial production by Tatua (Tatuani, New Zealand). It contains high concentrations of IGF and TGF.

They were administered to calves in the form of iso-nitrogen additives (12% protein), so that the biological effect can be attributed to the qualitative content of the whey fraction, and not to the amount of protein.

The volume of additives was gradually adapted to match the growth of rat pups (150 μl / 100 g body weight). Thus, the supplement provided from 1 to 5 mg of protein per day per calf.

The groups were as follows:

1) CTRL-w: CTRL cubs born to CTRL females that received water as an additive;

2) PR-w: PR cubs born to female PRs treated as supplements

water

3) PR-Lacprodan Di9224: young PRs born to female PRs treated with Lacprodan Di9224 as an additive;

4) PR-MSWP28: PR calves born to female PRs treated with MSWP28 as an additive;

6) PR - concentrate GF: young PR born to females of PR, receiving GF concentrate as an additive.

Two weeks after birth, at the time of sacrifice, the development of the nervous system in the young was evaluated in two ways: (i) using an approach that analyzes the expression of the target gene in the jejunum and (i) by measuring the contractile response of the ex-vivo jejunum to electrical stimulation field (EFS). In the latter method, the contractile response induced by the enteric nervous system is measured, and thus the maturation and degree of functioning of the enteric nervous system is measured.

The contractile response ex-vivo of the jejunum to acetylcholine was also evaluated, while the contractile response of the intestinal muscle proper was measured, which, therefore, reflects the maturation and degree of functioning of the intestinal muscle. The last experiment was performed as a control, which allowed the inventors to separate factors, i.e. nervous system and muscle controlling measurable contractile reactions.

Ex-vivo jejunum contractile response to EFS

To assess the maturation of the nervous system in a protein restriction model and the subsequent addition of sweet whey isolates in accordance with the invention, the jejunum contractile response to EFS was measured, which is an indicator of the role / ability of the neural network to stimulate muscle contraction. The possible role of any effect on muscular maturation of the intestine due to protein restriction or after addition of sweet whey isolates was evaluated by measuring the contractile reaction of the jejunum to acetylcholine.

The jejunum contractile response to EFS was reduced in PR-w compared to CTRL-w at a frequency of 10 Hz (P = 0.040) (Fig. 1). In PR-w compared with CTRL-w, the contractile reaction of the jejunum to acetylcholine did not significantly decrease, but only slightly (P = 0.181) (Figure 2). In a joint examination, the data reflect impaired neuromuscular interaction and, in particular, impaired maturation of the nervous system in the jejunum in pups with protein restriction.

The addition of sweet whey fractions (Lacprodan Di9224 and MSWP28) significantly increased (P = 0.007 and 0.026, respectively) the jejunum contractile response at 10 Hz to a level statistically similar to the controls (Fig. 1). This indicates the stimulating effect of sweet whey fractions on the ability to stimulate the nervous system in the jejunum, which reflects the development and functioning of the nervous system in the neonatal period. The maturation of the muscle compartment of the jejunum did not actually change significantly after the addition of sweet whey fractions (P = 0.199 and 0.828 for Lacprodan Di9224 and MSW P28, respectively), since the ability to contractile reaction of the jejunum to acetylcholine remained unchanged compared to PR-w (Fig. . 2).

A joint review of these data suggests that the standard and modified sweet whey protein (Lacprodan Di9224 and MSWP28, respectively, in the experiment) improve neuron-mediated muscular contractile response to EFS in the jejunum in PR pups. This suggests that the protein of sweet whey provides postnatal development and functionality of the enteric nervous system.

Such a stimulating effect was not observed in response to stimulation with acetylcholine, which suggests the absence of an improvement in the contractile response of smooth muscles to acetylcholine, i.e. lack of stimulation of postnatal smooth muscle maturation.

Thus, these data clearly show the beneficial effect of sweet whey protein on the development and functioning of the nervous system in the postnatal period. Based on these data, sweet whey represents a new nutritional solution for the prevention or treatment of disorders associated with immature and / or impaired enteric nervous system in young mammals up to the age of approximately six years, in particular during the neonatal period (for humans, usually defined as the first 6 months of life).

Expression of genes involved in the development of the intestinal nervous system

In the experiment on the rat model in Example 1, the effects of protein restriction as well as protein restriction, followed by the addition of milk fractions on the expression of the main genes involved in the biological pathways of the nervous system, were studied in the jejunum using an approach that included gene expression analysis. The genes studied are listed in Table 2, and the data are presented in Table 3. Data for genes expressed slightly or not at all are not expressed.

Among the remaining 35 genes expressed at the detection level, for 18 of them, a significant interaction was observed between the type of processing (protein restriction) and the additive (milk fraction) (table 3). Genes that are expressed with significant differences are genes that are primarily involved in the maturation or differentiation of neurons and glial cells, or are markers of the development and plasticity of the nervous system.

The authors of the present invention previously showed (Cettour and Faure, manuscript in preparation) that in protein-limited (PR) pups there are compensatory mechanisms that help to ensure the development of cells and the nervous system in the small intestine. These data confirm these results. In particular, the expression of genes for neurotrophic factors and their receptors, such as NGF (nerve growth factor), Gfral (receptor alpha-1 of the GDNF family), Gfra2 (receptor alpha-2 of the GDNF family), Ntf4 (neurotrophin 4), S100B (calcium binding protein B of the protein groups S100) and synaptophysin were significantly increased in the PR-w group compared to the CTRL-w group. This fact, apparently, reflects the imbalance and "stressful" situation in animals, which nevertheless remain characterized by a lack of stimulation of the maturation of the nervous system, as demonstrated by the results after stimulation of EFS.

Lacprodan Di9224 supplementation significantly increased the expression of the NGF, NGFr (nerve growth factor receptor) and Chrm3 (muscarinic receptor 3) genes and reduced the expression of S100b to a level similar to that of CTRL-w (table 3). The increased expression of the NGFr and NGF genes together with the increased expression of NGF in PR indicates activation of the growth, development and survival of neurons, as shown in Figure 1 [Tessarollo, (1998) Pleiotropic functions of neurotrophins in development. Cytokine Growth Factor Rev. 9 (2): 125-37]. It should be noted that the concentrations of NGF and NGFr were increased in PR-w compared to CTRL-w, but this increase was significant only for NGF.

MSWP28 supplementation significantly increased the expression of the Crhr1, ApoE, NGFr, Ntf3 (neurotrophin 3), Chrm2 and 3 (muscarinic receptor 2 and 3) and TGFb2 genes and significantly reduced the expression of S100b. Again, increased expression of the NGFr and Ntf3 genes may play a role in promoting the growth and development of neurons and may, at least in part, explain the neuronal maturation observed in FIG. 1.

Y, Kiank С, Stengel А. (2009) A role for corticotropin-releasing factor in functional gastrointestinal disorders Curr Gastroenterol Rep. Aug; 11(4):270-7; Vincelette J, Martin-McNulty B, Vergona R, Sullivan ME, Wang YX, (2006) Reduced cardiac functional reserve in apolipoprotein E knockout mice Transl Res. Jul; 148(1):30-6)].Muscarinic receptors encoded by the Chrm2 and Chrt3 gene are involved in contractility and intestinal motility in response to acetylcholine [Chen J, Wen J, Cai W. (2012) Smooth muscle adaptation and recovery of contractility after massive small bowel resection in rats. Exp Biol Med (Maywood). May 1; 237 (5): 578-84]. Similarly, the corticotropin releasing hormone receptor (Crhr), also known as the corticotropin releasing factor, and apolipoprotein E are involved in muscle contraction and motility [

Y, Kiank C, Stengel A. (2009) A role for corticotropin-releasing factor in functional gastrointestinal disorders Curr Gastroenterol Rep. Aug; 11 (4): 270-7; Vincelette J, Martin-McNulty B, Vergona R, Sullivan ME, Wang YX, (2006) Reduced cardiac functional reserve in apolipoprotein E knockout mice Transl Res. Jul; 148 (1): 30-6)].

After MSWP28 supplementation, chrm3 expression and expression of the previously mentioned genes involved in muscle contractility pathways were significantly increased compared to PR-w. This fact may indicate a stimulation of the maturation of the intestinal muscles, but it needs to be given additional attention, since increased gene expression did not translate into a measurable functional advantage, which is demonstrated by a constant contractile response to acetylcholine during killing of cubs (Fig. 2).

The addition of a growth factor concentrate significantly increased the expression of the Crhr1 gene and significantly reduced the expression of GDNF, GFra1, Neurod1, S100b, and Syp. Unlike MSWP 28 and Lacprodan Di 9224, no increased levels of expression of the NGF pathway (nerve growth factor pathway) were observed after the addition of growth factor concentrate. This is consistent with the result that no beneficial effect on the neuron-mediated contractile response (EFS) of the jejunum (Fig. 1) was observed after the addition of GF1.

The level of NGFr gene expression was significantly increased after the introduction of MSWP28 or LacprodanDi9224 additives, and increased gene expression induced by protein restriction in these groups receiving the additive was maintained. This clearly indicates a stimulation of the NGF pathway.

Neurotrophins like NGF and their receptors are expressed at a high level in the peripheral and central nervous system. In approaches with a targeted effect on the gene in mice, the role of neurotrophins in stimulating the evolutionary maturation of neurons of the peripheral and central nervous system was described, which confirms their decisive role in the development of the nervous system. A review is given in Tessarollo, L. (1998). NGF is also of great importance for the survival and preservation of sympathetic and sensitive neurons. Without it, these neurons undergo apoptosis [Freeman RS et al., (2004) NGF deprivation-induced gene expression: after ten years, where do we stand? "NGF and Related Molecules in Health and Disease." Prog. Brain Res. Progress in Brain Research 146: 111-26]. Nerve growth factor causes axon growth. There is evidence that NGF circulates throughout the body and is important in maintaining homeostasis. [Levi-Montalcini R (2004). "The nerve growth factor and the neuroscience chess board." Prog. Brain Res. 146: 525-7]. Human and cow milk also has NGF activity, which, presumably, plays a role in postnatal neurological development and has a neuroprotective effect in the newborn [Gaull, G.E., Wright, CE. & Isaacs, CE. (1985). Significance of growth modulators in human milk. Pediatrics. 75: 142-145].

The gene expression modifications shown in Table 3, and in particular, stimulation of the NGF pathway, probably contribute to the observed activation of neural stimulatory responses to EFS in the groups treated with MSW P28 and Lacprodan supplementation, as shown in FIG. 1.

Populations that may benefit from the invention include:

- premature infants, babies with low birth weight (<2500 g), babies with very low birth weight (<1500 g) and babies with extremely low birth weight (<1000 g);

- babies born prematurely or on time, with intrauterine growth retardation (IUGR), which occurred as a result of any adverse events during pregnancy;

- any newborn child and an infant with a delay in the development of the nervous system as a result of, for example, hypoxemia-ischemia at birth or any other undesirable phenomenon;

- any newborn child and infant or child up to the age of six years with disorders of the gastrointestinal tract (digestive disorders, motor disorders, gastrointestinal reflux, slow gastrointestinal transit, intolerance to oral feeding), Hirschsprung's disease and inflammation that covers the gastrointestinal tract intestinal tract (such as necrotizing enterocolitis), and obstructive pathological conditions.

The invention is further described with reference to the following examples. It should be understood that the invention in its claimed form should not be limited in any way by these examples.

Although the invention has been described by way of example, it should be understood that it is possible to make changes and modifications without departing from the scope of the invention defined by the claims. In addition, if equivalents of specific properties exist, such equivalents are included as if they were specifically mentioned in this description.

Example 1

Animal research (feeding and killing)

Animal experiments were carried out in accordance with permission No. 2120, issued by Office Vétérinaire Cantonal, Etat de Vaud. Two-month-old female Spreg-Dowley rats after one week of gestation were obtained from Harlan, Barcelona. On the day of their arrival, female rats were placed in individual cells and randomly assigned to a control group (CTRL) or a protein restriction group (PR). Animals had unlimited access to food and water, and they maintained a 12-hour light / dark cycle.

The diets of CTRL and PR females are detailed in Table 1. CTRL females received a control diet containing 20% protein (casein) in accordance with the standard requirement to provide rat protein during pregnancy (Reeves, PG, Nielsen, FH, Fahey, GC , JR. 1993. AIN-93 Purified Diets for Laboratory Rodents: Final Report of the American Institute of Nutrition Ad Hoc Writing Committee on the Reformulation of the AIN-76A Rodent Diet. J. Nutr. 123: 1939-1951). Female PRs received a PR diet containing 10% protein (casein). Both diets were isocaloric, with protein deficiency offset by the addition of corn starch.

CTRL and PR females received appropriate diets during pregnancy and lactation until the day of killing (14th day after birth (PND 14)).

In PND, 2 calves were randomly distributed between females of the same experimental group and the litter size was adjusted to 9 cubs per female, with a minimum number of males ranging from four to five per litter.

From PND 2 to PND 14, in the control or treatment groups, water or one of the following milk fractions was added to the food by hand / pipette daily. The volume of additives was gradually adapted to match the growth of rat pups (150 μl / 100 g body weight).

The groups and diets were as follows:

1) CTRL-w: CTRL cubs born to CTRL females that received water as an additive;

2) PR-w: PR cubs born to female PRs treated as supplements

water

3) PR-Lacprodan D19224: young PR born to female PR, received as an additive Lacprodan Di9224;

4) PR-MSWP28: PR calves born to female PRs treated with MSWP28 as an additive;

5) PR - GF concentrate: young PRs born to female PRs who received GF concentrate as an additive.

Milk fractions:

- Lacprodan Di9224: whey protein isolate, sweet whey, put into serial production by Arla Foods Ingredients, Denmark;

- MSWP28: sweet whey modified using the patented Nestle method (cGMP removal);

- GF concentrate: a growth factor concentrate launched into serial production by Tatua, New Zealand. It contains elevated levels of IGF and TGF.

On PND 14, a maximum of 10 CTRL and PR pups were weighed and then sacrificed by decapitation after halothane anesthesia.

The abdominal cavity was opened through a longitudinal section of the abdominal wall and the entire gastrointestinal tract was removed and freed from the mesentery. The first 1/3 of the jejunum segments were isolated after the Treitz ligament and divided into several segments. One segment was rinsed with phosphate buffered saline (PBS) at room temperature and placed in a cold Krebs solution to measure the contractile response ex-vivo. One segment was washed with ice-cold FBI to remove the cavity material, transferred to separate cryovials, instantly frozen in liquid nitrogen and stored at -80 ° C until gene analysis.

The development of neurons in the jejunum was evaluated in two ways: (i) using an approach with analysis of target gene expression, and (I) by measuring the ex-vivo contractile response of the jejunum to electric field stimulation (EFS).

Ex-vivo contractile reaction of the jejunum to electric field stimulation (EFS)

Segments of the distal jejunum about 1 cm long were placed in a cold Krebs solution (CaCl ₂ (5 mM), MgCl ₂ ⋅ 6H2O (1.2 mM), NaCl (120 mM), NaH ₂ PO ₄ ⋅H ₂ O (1, 2 mM), NaHCO ₃ (15.5 mM), KCl (5.9 mM), phenol red (0.005 mM) and glucose (11.5 mM)), 95% O ₂ /5% CO _{2 was} preoxygenated. Intestinal segments were suspended along their longitudinal axis in a tissue culture incubator (50 ml), filled with Krebs solution, oxygenated with 95% O ₂ /5% CO ₂ , at 37 ° C. One end of the muscle was attached to a fixed clamp. The other end was connected via an inelastic wire to an isometric force measurement sensor. Maintained for a stabilization period of 30 min to obtain spontaneous contractions. Then the fabric was stretched to the initial length so that any additional stretching would increase the tension at rest. Isometric contractility of the distal jejunum was induced by acetylcholine (Ach, 10 ^-5 M) or stimulation by electric field (EFS, 5-10 Hz). Ach was used for 1 min. After completing the dose-response curve, the bands were tested using 80 mM KCl to ensure that they retained their contraction ability. The signals were recorded digitally on a computer using Powerlab Chart 3.4. The results were normalized in cross-sectional area after careful blotting and weighing of tissue fragments. Nonlinear approximations were performed using the Prism 4.0 program (GraphPad Software, Inc., San Diego, USA). To obtain the frequencies, the power spectral density of the measured signal was analyzed. A high-pass filter with a cutoff frequency of 2 Hz was used (much less than the Nyquist frequency of 100 Hz). The Welch method was used to calculate the power spectral density from 1.3 seconds of overlap and superimposed with low-frequency sampling: a series of frequencies was set from 0 to 2 Hz with equal intervals of 0.05 Hz. The spectral power density calculations were performed using the Herzel algorithm. Three frequencies were selected corresponding to the three highest power values below 2 Hz.

Gene Expression Analysis

Total RNA was extracted from the jejunum of PR and CTRL cubs using the phenol / chloroform method with TriPure® reagent (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. Briefly, frozen tissue samples (50-100 mg) were homogenized in 1 ml of TriPure® using TissueLyser (Qiagen AG, Basel, Switzerland). The quantity and quality of the isolated RNA was measured using the RiboGreen RNA quantification kit (Invitrogen-Molecular Probes, Carlsbad, California, USA) and the RNA 6000 Nano LabChip kit (Agilent Technologies) in accordance with the instructions provided with these kits. Reverse transcription was performed using an oligoprimer (dT15) (Promega, Madison, Wisconsin, USA) and an ImProm-II (tm) reverse transcription system (Promega) in accordance with the manufacturer's instructions using 1 μg of total RNA.

Expression levels of the main genes involved in the development of the enteric nervous system (see Table 2) were evaluated by real-time 2-step quantitative RT-PCR using Sybergreen's RT Profiler PCR Array System (SABiosciences).

The measurements were carried out in duplicate using specific sets of TAMRA primers and fluorescent probes during the log-linear phase of the PCR reaction on a system for fast real-time PCR 7600НТ TaqMan Fast Real-Time PCR System (Applied Biosystems) using Sequence Detection Software version 2.2. PCR reactions were performed in 2 μl wells pre-loaded with specific primers and probes from the manufacturer into each of the 384 wells of the reaction card. The reaction was carried out using a final concentration of 0.8 ng / μl cDNA sample with TaqMan Universal PCR Master Mix (Applied Biosystems) universal PCR mixture containing AmpliTaq Gold DNA Polymerase DNA polymerase enzymes (Applied Biosystems), nucleotides and ROX fluorescent dye as a reference passive loading. The sequences of primers and probes used were designed and tested at Applied Biosystems and obtained from the rat library at Assay-on-Demand. The level of relative expression of each gene was normalized using geometric mean control genes (those whose expression was statistically stable for experimental groups in our conditions).

Statistical data

The effect of protein restriction was evaluated by comparing the PR and CTRL groups. The effect of the introduction of protein-based supplements was evaluated by comparing each PR group receiving the supplement with the PR-w group. Possible recovery to CTRL levels was evaluated by comparing each PR group receiving the supplement with the CTRL-w group.

Nonparametric methods were used to analyze the data; Wilcoxon's rank sum test was used to test differences between treatment groups. The Hodges-Lehman method was also used to calculate the paired difference between the types of treatment with a 95% confidence interval.

Statistical analysis of gene expression was performed on samples of primary cyclic thresholds (Ct), assuming that they are log 2-values. Univariate analysis of variance (ANOVA) was applied to the differences: the gene is the reference gene, the calculated p-value is the probability that at least one of the groups is different from the others. The calculations were performed using 5 potential constitutive genes or using the average of more than one; only the most stable ones were preserved according to the criteria of the least residual error.

Example 2

The following is an example of a baby formula composition for use in accordance with the present invention. This composition is for illustrative purposes only. The protein source is a mixture of 60% MSWP28 and 40% casein.

Claims (16)

1. The use of sweet whey protein in the prevention or treatment of at least one disorder associated with an immature or damaged enteric nervous system in an infant or young child, and the whey protein is administered to the infant or young child in a daily dose of 30 -80% of total protein intake. 2. The use according to claim 1, wherein the sweet whey protein is a modified sweet whey protein (MSWP) from which CGMP has been partially or completely removed. 3. The use according to claim 1 or 2, where the disorder is a violation of the motor function of the gastrointestinal tract, manifested in the form of slow intestinal transit, intestinal discomfort, hard stool, constipation and / or gastrointestinal reflux. 4. The use according to claim 1 or 2, where the pathological condition is a violation of the barrier function of the intestine, food intolerance or necrotizing enterocolitis. 5. The use according to claim 1 or 2, where the sweet whey protein contains at least 88 wt.% Protein, has a maximum fat content of 0.2 wt.% And a CGMP content of up to 40 wt.%. 6. The use of claim 2, wherein the sweet whey protein has a final CGMP content of less than 15 wt.%, Preferably less than 10 wt.%, More preferably less than 5 wt.%. 7. The use according to claim 1 or 2, where the protein of sweet whey has a degree of hydrolysis of from 2 to 20%. 8. The use according to claim 1 or 2, wherein the infant or young child is a premature or full-term infant, or a child under the age of six. 9. The use according to claim 1 or 2, where the infant or young child experienced hypoxemia-ischemia at birth, experienced or is experiencing IUGR, and / or had or is projected to have low, very low, or extremely low birth weight, and / or suffers or has suffered from a delay in the development of the enteric nervous system, either in utero, or during birth, or after birth. 10. The use according to claim 1 or 2, where the introduction of the infant or young child is carried out through a nursing mother and / or directly to the infant or young child. 11. The use according to claim 1 or 2, where the period of administration to an infant or young child has a duration of at least 4 weeks, preferably 2-12 months, and more preferably for a period of at least 18 months and even more, preferably until child age 6 years. 12. The use according to claim 1 or 2, where the protein of sweet whey is administered to an infant or a baby starting to walk, directly in pure form or diluted with water or breast milk, in the form of a biologically active food supplement (BAA), or together with milk fortifier, or with any milk supplement used during nutrition, in infant formula for premature babies, the initial infant formula or mixture for nursing infants, or infant formula for children 1-3 years old, or in a milk-based drink. 13. The use according to claim 1 or 2, where the introduction of a nursing mother or infant or young child is carried out orally, preferably by means of food, drinks, dietary supplements or pharmaceutical compositions. 14. The use according to claim 1 or 2, wherein the sweet whey protein is administered to a premature or full-term infant or young child at a dose of 1.6-3.2 g protein / 100 kcal, preferably 1.6-2.2 g protein / 100 kcal and even more preferably 1.8-2.1 g of protein / 100 kcal. 15. The use according to claim 1 or 2, wherein the sweet whey protein is administered in a composition comprising at least one prebiotic, preferably selected from inulin, fructooligosaccharide (FOS), short chain fructooligosaccharide (short chain FOS), galactooligosaccharide (GOS) and bovine oligosaccharides milk (CMOS) or breast milk oligosaccharides (HMOS). 16. The use according to claim 1 or 2, wherein the sweet whey protein is administered in a composition containing at least one probiotic, preferably selected from Bifidobacterium longum BB536 (ATCC BAA-999), Lactobacillus rhamnosus (CGMCC 1.3724), Bifidobacterium lactis (NCC281818 ) or mixtures thereof.

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